The innate immune response is the first line of defense against pathogens, and plays a critical role in antimicrobial defence. This response is initiated by host-encoded Pattern-Recognition Receptors (PRRs) that recognize evolutionarily conserved pathogen-derived signatures, known as Microbe-Associated Molecular Patterns (MAMPs), and activate MAMP-triggered immunity (MTI) (Boller & Felix, 2009). Furthermore, plants have evolved another strategy to perceive microbial pathogens through disease resistance (R) proteins, which recognize, directly or indirectly, divergent pathogen virulence determinants known as effector proteins, and establish effector-triggered immunity (ETI) (Jones & Dangl, 2006). Upon detection of MAMPs or pathogen effectors, plant cells rapidly induce a series of signalling events that involve for instance, the differential expression of short interfering RNAs (siRNAs) and microRNAs (miRNAs) (Pumplin N & Voinnet O, 2013). Recently, several siRNAs and miRNAs were found to orchestrate MTI and ETI responses (Weiberg et al., 2014), implying a key role of RNA silencing in the regulation of the plant immune system.
RNA silencing is an ancestral gene regulatory mechanism that controls gene expression at the transcriptional (TGS, Transcriptional Gene Silencing) and post-transcriptional (PTGS, Post-transcriptional Gene Silencing) levels. In plants, this mechanism has been initially characterized in antiviral resistance (Hamilton & Baulcombe, 1999). The core mechanism of RNA silencing starts with the production of double stranded RNAs (dsRNAs) that are processed by RNase-III enzymes DICERs into 20-24 nt small RNA duplexes that subsequently associate with an Argonaute (AGO) protein, which represent the central component of the RNA-induced silencing complexes (RISC). One strand, the guide, remains bound to the AGO effector protein to regulate genes in a mature RISC, while the other strand, the passenger, is degraded. The plant model Arabidopsis thaliana encodes 4 DICER-like proteins and 10 AGOs. DCL1 processes miRNA precursors into miRNA/miRNA* duplexes and the guide miRNA strand directs AGO1-RISC to sequence complementary mRNA targets to trigger their degradation and/or translation inhibition. DCL2 and DCL4 are involved in the biogenesis of short interfering RNAs (siRNAs) derived from viral dsRNAs and play a critical role in antiviral resistance (Deleris et al., 2006; Diaz-Pendon et al., 2007). These DICER-like proteins are also involved, together with DCL3, in the production of siRNAs derived from transposable elements, read through, convergent or overlapping transcription, endogenous hairpins as well as some miRNA precursors (Bologna & Voinnet, 2014). In addition, a large proportion of dsRNAs are produced by RNA-dependent RNA polymerases (RDRs) that convert single stranded RNAs into dsRNAs. RDR6, which is one out of six Arabidopsis RDRs, produces dsRNAs from viral, transgene transcripts as well as some endogenous transcripts including transposable elements (Mourrain et al., 2000; Dalmay et al, 2000.; Schwach et al., 2005; Xie et al., 2004). These dsRNAs are processed in part by DCL4 and DCL2 into 21 nt and 22 nt siRNAs, respectively, which direct PTGS of endogenous sequence complementary mRNA targets or exogenous RNAs derived from sense-transgenes or viral RNAs (Bologna & Voinnet, 2014). Furthermore, both siRNA and miRNA duplexes are methylated by the small RNA methyltransferase HEN1 and this modification is essential for their stability (Yu et al, 2005; Li et al., 2005; Chen et al., 2012).
Although endogenous miRNAs and siRNAs were initially characterized in various plant development processes, they have more recently emerged as key regulators of the plant innate immune response (Pumplin & Voinnet, 2013). For instance, the miR393 is a conserved microRNA that is induced during MTI and that contributes to antibacterial resistance in Arabidopsis (Navarro et al., 2006, 2008; Fahlgren et al., 2007; Li et al., 2010). Furthermore, phenotypical analyses in mutants that are defective in PTGS exhibit enhanced disease susceptibility to fungal, bacterial and oomycete pathogens (Navarro et al., 2008; Qiao et al., 2013; Ellendorff U et al., 2009; Navarro & Voinnet, 2008 WO/2008/087562), supporting a central role of this gene regulatory process in resistance against unrelated pathogens. However, despite the well-established role of RNA silencing in resistance against viral and non-viral pathogens, very little is known on the physiological relevance, or on the activity, of RNA silencing in tissues that are relevant for the entry and/or propagation of phytopathogens.
Phytopathogenic microbes can be divided into biotrophs, hemibiotrophs and necrotrophs according to their different lifestyles. Biotrophic pathogens can take-up nutrients from living host cells and maintain host cell viability, while necrotrophs kill host cells and feed on dead tissues. Hemibiotrophs use an early biotrophic phase followed by a necrotrophic phase. These phytopathogenic microbes use different strategies to enter inside host tissues. The majority of fungal and oomycete pathogens first produce spores, which adhere to the plant surface and further germinate to form a germ tube. Subsequently, the germ tube develops an appressorium that can perforate, through a penetration peg, the cuticle and cell wall layer through mechanical forces (Horbach et al., 2011) and therefore enter inside host tissues. Once inside plant tissues, the hyphal tip forms a second specialized structure referred to as the haustorium that can uptake nutrients from host cells but also represents a major site of pathogen effector secretion (Mendgen & Hahn, 2002; Horbach et al., 2011). Other pathogens do not perforate the cuticle cell wall layer but instead use natural openings to reach internal host tissues. For instance, hemibiotrophic bacterial pathogens such as Pseudomonas and Xanthomonas use hydathodes, stomata or woundings as natural entry sites for their endophytic colonization (Dou & Zhou, 2012). These bacterial pathogens can also enter plant tissues through the base of trichomes in some instances (Xin & He, 2013). Some fungal pathogens can also use natural openings to enter plant tissues. For example, Cladosporium fulvum colonizes internal host tissues through stomatal openings by forming long, branched intercellular hyphal structures with no obvious haustorium (Thomma et al., 2005). It is therefore not surprising that plants have evolved sensitive pathogen recognition mechanisms, and sophisticated defense responses, in these tissues/cell types to prevent pathogen entry (Melotto et al., 2006; Hugouvieux et al., 1998). As an example, plants can perceive bacterial MAMPs at the level of guard cells and in turn trigger stomatal closure, which efficiently controls the access of bacterial pathogens to internal host tissues (Melotto et al., 2006). Active defense responses have also been reported at hydathodes and at the base of trichomes (Hugouvieux et al., 1998; Yu et al., 2013; Frerigmann et al., 2012; Jakoby et al., 2008), although very little is known on the detailed mechanisms that orchestrate such local innate immune responses.
Once inside the intercellular space of plant leaves, biotrophic and hemibiotrophic pathogens use different strategies to colonize distal plant tissues. For example, RNA viruses use alive phloem tissues as a major route to propagate in systemic tissues and this viral spreading is restricted by PTGS. As an example, Arabidopsis mutants that are defective in the two major antiviral Dicer-like 2 (DCL2) and DCL4 exhibit long distance propagation of several RNA viruses (Deleris et al., 2006; Diaz-Pendon et al., 2007). By contrast, fungal, oomycete and bacterial pathogens can spread in distal plant tissues through xylem vessels, which are dead cells that transport nutrients and water from the root to the aerial part of the plant. The plant defense responses that restrict vascular spreading involve multiple processes including, for instance, the production of reactive oxygen species (ROS) and antimicrobial proteins from adjacent xylem parenchyma cells, the plant-induced physical obstruction of vascular vessels through lignification, the efficient recognition of pathogen effectors through R proteins (Guy et al., 2013). However, it is not known whether PTGS plays any role in restricting the propagation of such non-viral pathogens within and around xylem vessels.
In the present invention, the inventors first provide experimental evidence that PTGS plays a critical role in controlling the entry of a virulent Pseudomonas syringae strain at the level of hydathodes and stomata in Arabidopsis thaliana. Furthermore, they show that PTGS efficiently restricts vascular propagation of this pathogenic bacterium in Arabidopsis leaf vasculature. Based on these observations, they have selected the most relevant promoters that are active enough in dedicated tissues or cell types to generate a whole series of visual inverted repeat PTGS reporter lines in the model plant Arabidopsis thaliana. These PTGS reporter lines are based on the tissue-specific silencing of an endogenous gene involved in chlorophyll biosynthesis and therefore allow an easy monitoring of PTGS activity in tissues/cell types that are relevant for pathogen entry, propagation or replication, namely the hydathodes, guard cells, epidermal cells, xylem parenchyma cells, cells at the base of trichomes, phloem companion cells and mesophyll cells.
The intended applications therefore deal with methods to increase disease resistance in cultivated plants by enhancing PTGS activity in tissues/cell types that are relevant for pathogen entry, propagation or replication. For instance, they can be used for high-throughput screening of natural or synthetic compounds that can enhance the chlorotic phenotype and likewise promote PTGS in tissues/cell types that are physiologically relevant for plant disease management. Given the critical and widespread role of PTGS in plant disease resistance, the identified PTGS inducer compounds will likely enhance resistance against unrelated pathogens in Arabidopsis but also in agriculturally important crops by preventing pathogen entry, propagation and/or replication. As a proof-of-concept experiment, the inventors have challenged these PTGS sensors with commercially available natural or synthetic—active or formulated—compounds that are known to increase disease resistance in cultivated plants. Results from these analyses indicated that well-characterized compounds can indeed promote PTGS on one specific silencing reporter. Besides providing novel insights into the mode of action of these commercially available molecules, these experiments indicate that such PTGS reporters can be exploited to conduct high-throughput screening in order to identify novel natural/synthetic compounds, microorganisms or extracts from micro- or macro-organisms that have the potential to promote disease resistance in cultivated plants by enhancing PTGS activity. Furthermore, such reporter system can also be used to elucidate the mode of action of compounds, microorganisms or extracts from micro- or macro-organisms with known antimicrobial activity in order to meet the requirement of the legislation for their commercialization.